The long-term objective of the this research is to develop a gene transfer system for the N2-fixing actinomycete Frankia. However, aspects of Frankia growth and genetics present challenges to the development of gene transfer methods. Among the potential barriers is the slow growth rate of Frankia in laboratory culture. To compound the problem, strains such as strain CpI1, often form tightly packed mycelial masses (pellets) in liquid culture. Pellet morphology may deny sub-populations of cells within the pellet access to nutrients and subsequently limit growth. Pellet formation may also inhibit gene transfer in Frankia by limiting the number of viable target cells available, or by suppressing the growth of genetically altered mycelia. The immediate goal is to clarify and improve on the physiological state of laboratory cultures of Frankia. The results of preliminary growth studies have shown that the anionic polymer, Carbopol 941 (carbopol; BF Goodrich, Cleveland, OH) encourages dispersed growth of Frankia in liquid culture. The PI will investigate the effects of carbopol concentration on the growth rate and morphology of Frankia and, concurrently, test the usefulness of the fluorescent cell impermeant dye Sytox Green (SG; Molecular Probes, Eugene, OR) in determining Frankia viability. SG selectively stains dead or dying cells on the basis of cell membrane integrity. Because the condition of the cell membrane impacts cellular processes, cell viability can be inferred from the staining patterns with SG. The viability of Frankia mycelia and differentiated vesicles growing in the presence and absence of carbopol will be assessed using SG and epifluorescence microscopy. The rationale for this work is that if carbopol can lengthen the exponential growth phase and facilitate dispersed growth, its use in media to prepare recipient cells, or to grow genetically manipulated cells, may increase the potential for introduction of DNA into Frankia, and subsequent expression of recombinant traits.
